Catecholamine derivatives for obesity and neurological disorders

ABSTRACT

Novel compounds, compositions, and methods related to the activation of the TrkB receptor are provided. The methods include administering in vivo or in vitro a therapeutically effective amount of a compound containing a catecholamine backbone and pharmaceutically acceptable salts, prodrugs, and derivatives thereof. Specifically, methods, compositions, and compounds for the treatment of disorders including neurological disorders, neuropsychiatric disorders, and metabolic disorders are provided. For example, a first method is provided of treating or reducing the risk of depression, anxiety, or obesity in a subject, which includes administering to the subject a therapeutically effective amount of the described compounds. A further method of promoting neuroprotection in a subject also is provided, which includes administering to the subject a therapeutically effective amount of the described compounds.

CROSS-REFERENCE TO PRIORITY APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/161,911, filed Mar. 20, 2009, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support from the NationalInstitutes of Health Grant number R01-CA127119. The government hascertain rights in this invention.

BACKGROUND

Neurologic and neuropsychiatric disorders such as depression, anxiety,amyotrophic lateral sclerosis, and central nervous system injuries, toname a few, afflict millions of people every year and result in amultitude of symptoms including weight change, decreased energy,headaches, digestive problems, chronic pain, paralysis, and in certaininstances, death. Neurotrophins, such as brain-derived neurotrophicfactor (BDNF), affect various neuronal populations involved inneurologic, neuropsychiatric, and metabolic disorders. Neurotrophinslike BDNF are natural ligands of tyrosine kinase receptor TrkB. TrkB isactivated in hippocampal neurons prior to BDNF expression bynorepinephrine, which is a type of catecholamine.

SUMMARY

Novel compounds and methods for the treatment of disorders includingneurological disorders, neuropsychiatric disorders (e.g., depression oranxiety), and metabolic disorders (e.g., obesity) are provided. Themethods include administering to a subject a therapeutically effectiveamount of a compound having the following formula:

or a pharmaceutically acceptable salt or prodrug thereof. In thiscompound, R¹ is hydrogen, —OH, or ═O; R² is hydrogen, substituted orunsubstituted C₁₋₄ alkyl, substituted or unsubstituted C₁₋₄ heteroalkyl,substituted or unsubstituted C₂₋₄ alkenyl, substituted or unsubstitutedC₂₋₄ heteroalkenyl, substituted or unsubstituted C₂₋₄ alkynyl,substituted or unsubstituted C₂₋₄ heteroalkynyl, or substituted orunsubstituted carbonyl; R³ and R⁴ are each independently selected fromhydrogen, substituted or unsubstituted C₁₋₁₂ alkyl, substituted orunsubstituted C₃₋₁₂ cycloalkyl, substituted or unsubstituted C₁₋₁₂heteroalkyl, substituted or unsubstituted C₂₋₁₂ alkenyl, substituted orunsubstituted C₃₋₁₂ cycloalkenyl, substituted or unsubstituted C₂₋₁₂heteroalkenyl, substituted or unsubstituted C₂₋₁₂ alkynyl, substitutedor unsubstituted C₃₋₁₂ cycloalkynyl, substituted or unsubstituted C₂₋₁₂heteroalkynyl, substituted or unsubstituted carbonyl; and R⁵, R⁶, and R⁷are each independently selected from hydrogen, halogen, —OH, or alkoxy,wherein one of R¹, R², R³, R⁴, R⁵, R⁶, or R⁷ is not hydrogen; wherein ifone of R³ or R⁴ is a saccharide, R⁵ is not hydrogen; wherein if R¹ is—OH, one of R², R³, R⁴, R⁵, R⁶, or R⁷ is not hydrogen; if R¹ is —OH andone of R³ or R⁴ is —CH₃, one of R², R³, R⁴, R⁵, R⁶, or R⁷ is nothydrogen; if R² is —COOH, one of R¹, R³, R⁴, R⁵, R⁶, or R⁷ is nothydrogen; and if R² is —COOH and R¹ is —OH, then one of R³, R⁴, R⁵, R⁶,or R⁷ is not hydrogen. In some examples, if R¹ is —OH and one of R³ orR⁴ is isopropyl, one of R², R³, R⁴, R⁵, R⁶, or R⁷ is not hydrogen.

A method for the treatment of disorders including neurologicaldisorders, neuropsychiatric disorders, and metabolic disorders usingthese compounds is related to treating or reducing the risk ofdepression, anxiety, or obesity in a subject, which includesadministering to the subject a therapeutically effective amount of thecompound described above or a derivative thereof. A method of promotingneuroprotection in a subject also is provided, which includesadministering to the subject a therapeutically effective amount of thecompound described above or a derivative thereof.

A method of activating a TrkB receptor on a neuron also is provided. Themethod includes providing a neuron having a TrkB receptor, andcontacting the TrkB receptor in vitro or in vivo with a compound asdescribed above or a derivative thereof in an amount sufficient toactivate the TrkB receptor. The neuron can be, for example, a mammaliancell.

Also provided herein are novel compositions including the compoundsdescribed herein or derivatives thereof and an anti-depressant or ananti-anxiolytic. Methods of making the compounds described herein arealso provided in which L-DOPA or L-DOPS are used as starting materials.

The details of one or more examples of the compounds, compositions, andmethods are set forth in the accompanying drawings and the descriptionbelow. Other features, objects, and advantages will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the chemical structures of several catecholamines.

FIG. 2 shows immunofluorescent staining images showing thatcatecholamines activate TrkB in primary hippocampal neurons.

FIG. 3 shows Western blots demonstrating that catecholamines activateTrkB in primary cortical neurons.

FIG. 4 shows Western blots demonstrating that K252a blockscatecholamines' stimulatory effect on TrkB activation in corticalneurons.

FIG. 5 shows Western blots illustrating the phosphorylation of TrkB incells treated with increasing concentrations of dopamine,norepinephrine, and epinephrine.

FIG. 6 shows Western blots illustrating the stimulatory effect of 100 nMnorepinephrine treatment on TrkB phosphorylation in cortical neurons forincreasing amounts of time.

FIGS. 7A and 7B show the results of an in vitro binding assay withpurified TrkB and TrkA extracellular domain (ECD) or intracellulardomain (ICD) (2 μg) and ³H-catecholamines and ³H-7,8-dihydroxyflavone.FIG. 7A illustrates that the catecholamines selectively bind the ICD butnot the ECD of TrkB receptor, and 7,8-dihydroxylflavone binds the ECDbut not the ICD of TrkB receptor. FIG. 7B includes a Scatchard plotanalysis of these data indicating the binding constants of thecatecholamines.

FIG. 8 shows Western blots illustrating that catecholamines cause TrkBdimerization.

FIG. 9 shows Western blots illustrating that catecholamines induce TrkBautophosphorylation.

FIG. 10 shows Western blots illustrating that catecholamines activateTrkB in the presence of BDNF antibody in primary cortical neurons.

FIG. 11 shows Western blots indicating that NT-3 or NT-4 antibody isunable to block the TrkB agonistic activity of catecholamines.

FIG. 12 shows Western blots demonstrating that catecholamines activateTrkB in BDNF null cortical neurons.

FIG. 13 shows Western blots illustrating that norepinephrine antibodydiminishes the TrkB agonistic activity by norepinephrine in corticalneurons.

FIG. 14 shows Western blots demonstrating that catecholamines activateTrkB in a catecholamine receptor independent manner.

FIGS. 15A and 15B show Western blots indicating that catecholaminesselectively activate TrkB receptors in cortical neurons. In FIG. 15A,catecholamines activated TrkB in wild-type but not TrkB-null neurons. InFIG. 15B, catecholamines induced TrkB activation in both wild-type andTrkC knockout neurons.

FIG. 16 shows Western blots illustrating that catecholamines activatecortical neurons with a TrkB F616A mutant. Catecholamines-provoked TrkBphosphorylation was selectively blocked by 1NMPP1 but not K252a, whileserotonin had no effect (left panel). Catecholamines-induced TrkBactivation was robustly inhibited by K252a but not by 1NMPP1 in TrkAF592A neurons (right panel).

FIG. 17 shows Western blots demonstrating that dopamine transporter andnorepinephrine transporter inhibitors block TrkB activation bycatecholamines in primary cortical neurons.

FIG. 18 shows Western blots illustrating that a combination of dopaminetransporter and norepinephrine transporter inhibitors suppress TrkBactivation by catecholamines.

FIG. 19 shows Western blots indicating that norepinephrine transporterdepletion blocks TrkB activation by norepinephrine and epinephrine.

FIG. 20 shows Western blots indicating that norepinephrine antibodyselectively blocks the agonistic effect of norepinephrine but notdopamine or epinephrine.

FIG. 21 shows Western blots indicating that dopamine and adrenergicreceptor antagonists do not block the agonistic effect of catecholamineon TrkB.

FIG. 22A shows that OCT3 inhibitor has no effect on TrkB activation bycatecholamines.

FIG. 22B shows that EDTA blocks the stimulatory effect on TrkBactivation by Zn but not catecholamine.

FIG. 23 shows Western blots illustrating that catecholamine derivativesactivate TrkB in primary cortical neurons.

FIGS. 24A, 24B, and 24C show Western blots illustrating thatcatecholamine derivatives (Compound I-11 and Compound I-8) activate TrkBorally. FIG. 24A shows the immunoblot results for Compound I-11. FIG.24B shows the immunoblot results for Compound I-8. FIG. 24C shows theimmunoblot results for Compound I-2.

FIGS. 25A and 25B show Western blots illustrating that catecholaminederivatives activated TrkB in a dose-dependent manner. FIG. 25A showsthe immunoblot results for Compound I-11. FIG. 25B shows the immunoblotresults for Compound I-8.

FIGS. 26A and 26B show Western blots illustrating that catecholaminederivatives activate TrkB in primary cortical neurons.

FIGS. 27A, 27B, and 27C show Western blots illustrating thatcatecholamine derivatives (Compound I-11 and Compound I-8) are orallybioavailable. FIG. 27A shows the immunoblot results for oraladministration. FIG. 27B shows the immunoblot results forintraperitoneal injection. FIG. 27C shows the immunoblot results for thecompounds dissolved in drinking water.

FIGS. 28A and 28B show graphs and Western blots illustrating thatCompound I-11 and Compound I-8 suppress neuronal apoptosis in a TrkBdependent manner. FIG. 28A shows that norepinephrine blocksglutamate-provoked neuronal cell death in a TrkB dependent manner. FIG.28B shows that Compounds I-11 and 1-8 protect neurons from KA-inducedapoptosis. FIG. 28C shows that 1NMPP1, but not K252a, blocks TrkBactivation by Compounds I-11 and I-8.

FIGS. 29A and 29B show graphs illustrating the forced swim test resultsof mice treated with Compound I-11 and Compound I-8. FIG. 29A shows thatthe swimming immobility of mice treated with Compounds I-11 and 1-8 wasdecreased. FIG. 29B shows the swimming immobility of TrkB F616 knockinmice treated with Compounds I-11 and 1-8 after pretreatment with salineor 1NMPP1.

DETAILED DESCRIPTION

Described herein are compounds, compositions, and methods for theactivation of a TrkB receptor. These compounds, compositions, andmethods are effective in the treatment of diseases and illnessesassociated with the activation of the TrkB receptor includingneurological disorders, neuropsychiatric disorders, and metabolicdisorders. Examples of neurological disorders include depression,anxiety, Alzheimer's disease, central nervous system (CNS) injuries, andthe like. Examples of metabolic disorders include obesity andhyperphagia. Specifically, provided herein are compounds containing acatecholamine backbone and pharmaceutically acceptable salts, prodrugs,and derivatives thereof. Methods of their use in the treatment ofneurological disorders, neuropsychiatric disorders, and obesity are alsodescribed herein.

The compounds containing a catecholamine backbone as described hereinare represented by Compound I:

and pharmaceutically acceptable salts and prodrugs thereof.

In Compound I, R¹ is hydrogen, —OH, or ═O.

Also, in Compound I, R² is hydrogen, substituted or unsubstituted C₁₋₄alkyl, substituted or unsubstituted C₁₋₄ heteroalkyl, substituted orunsubstituted C₂₋₄ alkenyl, substituted or unsubstituted C₂₋₄heteroalkenyl, substituted or unsubstituted C₂₋₄ alkynyl, substituted orunsubstituted C₂₋₄ heteroalkynyl, or substituted or unsubstitutedcarbonyl. R² can be, for example,

wherein R⁸ and R⁹ are each independently selected from hydrogen,substituted or unsubstituted C₁₋₄ alkyl, substituted or unsubstitutedC₂₋₄ alkenyl, or substituted or unsubstituted C₂₋₄ alkynyl. As anexample, R² can be

Additionally, in Compound I, R³ and R⁴ are each independently selectedfrom hydrogen, substituted or unsubstituted C₁₋₁₂ alkyl, substituted orunsubstituted C₃₋₁₂ cycloalkyl, substituted or unsubstituted C₁₋₁₂heteroalkyl, substituted or unsubstituted C₂₋₁₂ alkenyl, substituted orunsubstituted C₃₋₁₂ cycloalkenyl, substituted or unsubstituted C₂₋₁₂heteroalkenyl, substituted or unsubstituted C₂₋₁₂ alkynyl, substitutedor unsubstituted C₃₋₁₂ cycloalkynyl, substituted or unsubstituted C₂₋₁₂heteroalkynyl, substituted or unsubstituted carbonyl.

Further, in Compound I, R⁵, R⁶, and R⁷ are each independently selectedfrom hydrogen, halogen, —OH, or alkoxy. R⁵, R⁶, and R⁷ can each be, forexample, fluorine.

In Compound I, one of R¹, R², R³, R⁴, R⁵, R⁶, or R⁷ is not hydrogen.

Also, in Compound I, if one of R³ or R⁴ is a saccharide, R⁵ is nothydrogen.

Additionally, in Compound I, if R¹ is —OH, one of R², R³, R⁴, R⁵, R⁶, orR⁷ is not hydrogen, or if R¹ is —OH and one of R³ or R⁴ is —CH₃, one ofR², R³, R⁴, R⁵, R⁶, or R⁷ is not hydrogen. In some examples, if R¹ is—OH and one of R³ or R⁴ is isopropyl, one of R², R³, R⁴, R⁵, R⁶, or R⁷is not hydrogen.

Further, in Compound I, if R² is —COOH, one of R¹, R³, R⁴, R⁵, R⁶, or R⁷is not hydrogen, or if R² is —COOH and R¹ is —OH, then one of R³, R⁴,R⁵, R⁶, or R⁷ is not hydrogen.

In Compound I, R² and NR⁴ can combine to form a substituted orunsubstituted heterocycloalkyl or a substituted or unsubstitutedheteroaryl. In the combination of R² and NR⁴, the substituted orunsubstituted heteroaryl is other than a pyrrole. For example, R² can ben-butane and NR⁴ can be methanamine that combine to form a piperidine.Similarly, R³ and NR⁴ can combine to form a substituted or unsubstitutedheterocycloalkyl or a substituted or unsubstituted heteroaryl.

Specific examples of Compound I are as follows:

The compounds described herein can be prepared in a variety of waysknown to one skilled in the art of organic synthesis or variationsthereon as appreciated by those skilled in the art. The compoundsdescribed herein can be prepared from readily available startingmaterials. Optimum reaction conditions may vary with the particularreactants or solvents used, but such conditions can be determined by oneskilled in the art.

Variations on Compound I include the addition, subtraction, or movementof the various constituents as described for each compound. Similarly,when one or more chiral centers is present in a molecule, the chiralityof the molecule can be changed. Additionally, compound synthesis caninvolve the protection and deprotection of various chemical groups. Theuse of protection and deprotection, and the selection of appropriateprotecting groups can be determined by one skilled in the art. Thechemistry of protecting groups can be found, for example, in Wuts andGreene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons,2006, which is incorporated herein by reference in its entirety.

As used herein, the terms alkyl, alkenyl, and alkynyl include straight-and branched-chain monovalent substituents. Examples include methyl,ethyl, isobutyl, 3-butynyl, and the like. Heteroalkyl, heteroalkenyl,and heteroalkynyl are similarly defined but may contain O, S, or Nheteroatoms or combinations thereof within the backbone. The termsubstituted indicates the main substituent has attached to it one ormore additional components, such as, for example, OH, halogen, or one ofthe substituents listed above.

Reactions to produce the compounds described herein can be carried outin solvents, which can be selected by one of skill in the art of organicsynthesis. Solvents can be substantially nonreactive with the startingmaterials (reactants), the intermediates, or products under theconditions at which the reactions are carried out, i.e., temperature andpressure. Reactions can be carried out in one solvent or a mixture ofmore than one solvent. Product or intermediate formation can bemonitored according to any suitable method known in the art. Forexample, product formation can be monitored by spectroscopic means, suchas nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C), infraredspectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high performance liquidchromatograpy (HPLC) or thin layer chromatography.

The compounds described by Compound I and pharmaceutically acceptablesalts and prodrugs thereof can be made, for example, usingL-dihydroxyphenylserine (L-DOPS) or N-protected L-DOPS as a startingmaterial. A method of making L-DOPS is shown in Scheme 1.

L-DOPS is shown as Compound B in Scheme 1 and N-protected L-DOPS isshown as Compound A in Scheme 1. Examples of compounds that can be madefrom L-DOPS and N-protected L-DOPS are shown in Scheme 2.

Another example of a method of making a compound described by Compound Iand pharmaceutically acceptable salts and prodrugs thereof includesusing L-dihydroxyphenylalanine (L-DOPA) as the starting material. Anexample of a method of making a compound described by Compound I fromL-DOPA is shown in Scheme 3.

A further example of a method of making a compound described by CompoundI and pharmaceutically acceptable salts and prodrugs thereof from L-DOPAis shown in Scheme 4.

The compounds described herein by Compound I can be methylated at theprimary amine to form N-methylated derivatives as shown in Scheme 5.

The methods described herein include a method of treating or reducingthe risk of disorders associated with activation of the TrkB receptorincluding neurological disorders, neuropsychiatric disorders, andmetabolic disorders in a subject by administering to the subject atherapeutically effective amount of Compound I or a derivative thereof.Examples of neurological and neuropsychiatric disorders includedepression, anxiety, Alzheimer's disease, central nervous system (CNS)injuries, and the like. Examples of metabolic disorders include obesityand hyperphagia. This method optionally includes the step of selecting asubject with or at risk of developing the neurological disorder,neuropsychiatric disorder, or metabolic disorder (e.g., obesity).Compound I or a derivative thereof can be administered systemically(e.g., orally, parenterally (e.g., intravenously), intramuscularly,intraperitoneally, transdermally (e.g., by a patch), extracorporeally,topically, by inhalation, subcutaneously or the like), by administrationinto the central nervous system (e.g., into the brain (intracerebrallyor intraventricularly), spinal cord, or into the cerebrospinal fluid),or any combination thereof.

Also provided is a method of promoting neuroprotection in a subject byadministering to the subject a therapeutically effective amount ofCompound I or derivative thereof as described herein. This methodoptionally includes the step of selecting a subject in need ofneuroprotection. A subject in need of neuroprotection can, for example,be a subject that has or is at risk of developing a central nervoussystem disease (e.g., amyotrophic lateral sclerosis (ALS)) or a centralnervous system injury. A central nervous system injury includes, forexample, a brain injury, a spinal cord injury, a cerebrovascular event(e.g., a stroke), or a central nervous system surgery. Neuroprotectionincludes protecting neurons after injury or disease has occurred orbefore the onset of disease or injury.

The methods can further comprise testing the effectiveness of Compound Ior derivative thereof as described herein. Testing the effectiveness caninclude, but is not limited to, imaging (e.g., Magnetic ResonanceImaging (MRI)) and functional measurements (e.g., survival or clinicalsymptoms like analysis of speech patterns, logic, comprehension, memory,mood, and orientation). The method optimally further comprises adjustingthe dosage or treatment regimen of Compound I or derivative thereof asdescribed herein.

Further provided is a method of activating a TrkB receptor on a neuron(e.g., a mammalian cell). This method includes the steps of providing aneuron having a TrkB receptor and contacting the TrkB receptor in vitroor in vivo with Compound I or a derivative thereof as described hereinin an amount sufficient to activate the TrkB receptor. Also provided isa method of screening for an agent that potentiates TrkB receptoractivation. The screening method includes activating the TrkB receptoron a neuron as described and contacting the neuron with the agent to bescreened. An enhanced effect indicates the agent potentiates the effectof Compound I or derivative thereof as described herein.

The compounds described herein or derivatives thereof can be provided ina pharmaceutical composition. Depending on the intended mode ofadministration, the pharmaceutical composition can be in the form ofsolid, semi-solid or liquid dosage forms, such as, for example, tablets,suppositories, pills, capsules, powders, liquids, or suspensions,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions include a therapeutically effectiveamount of the compounds described herein or derivatives thereof incombination with a pharmaceutically acceptable carrier and, in addition,may include other medicinal agents, pharmaceutical agents, carriers, ordiluents. By pharmaceutically acceptable is meant a material that is notbiologically or otherwise undesirable, which can be administered to anindividual along with the selected compound without causing significantunacceptable biological effects or interacting in a deleterious mannerwith the other components of the pharmaceutical composition in which itis contained.

As used herein, the term carrier encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, orother material well known in the art for use in pharmaceuticalformulations. The choice of a carrier for use in a composition willdepend upon the intended route of administration for the composition.The preparation of pharmaceutically acceptable carriers and formulationscontaining these materials is described in, e.g., Remington: The Scienceand Practice of Pharmacy, 21st Edition, ed. University of the Sciencesin Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005.Examples of physiologically acceptable carriers include buffers such asphosphate buffers, citrate buffer, and buffers with other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol(PEG), and PLURONICS™ (BASF; Florham Park, N.J.).

Compositions containing Compound I or derivative thereof as describedherein suitable for parenteral injection may comprise physiologicallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, and sterile powders for reconstitution intosterile injectable solutions or dispersions. Examples of suitableaqueous and nonaqueous carriers, diluents, solvents or vehicles includewater, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol,and the like), suitable mixtures thereof, vegetable oils (such as oliveoil) and injectable organic esters such as ethyl oleate. Proper fluiditycan be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be promoted by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Isotonic agents, for example, sugars, sodium chloride, and thelike may also be included. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration of Compound I or derivativethereof as described herein include capsules, tablets, pills, powders,and granules. In such solid dosage forms, the compounds described hereinor derivatives thereof is admixed with at least one inert customaryexcipient (or carrier) such as sodium citrate or dicalcium phosphate or(a) fillers or extenders, as for example, starches, lactose, sucrose,glucose, mannitol, and silicic acid, (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example, paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol, and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others known in the art. They may contain opacifying agentsand can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions that can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration of Compound I or derivativethereof as described herein include pharmaceutically acceptableemulsions, solutions, suspensions, syrups, and elixirs. In addition tothe active compounds, the liquid dosage forms may contain inert diluentscommonly used in the art, such as water or other solvents, solubilizingagents, and emulsifiers, as for example, ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils,in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil,castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol,polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures ofthese substances, and the like.

Besides such inert diluents, the composition can also include additionalagents, such as wetting, emulsifying, suspending, sweetening, flavoring,or perfuming agents.

Suspensions, in addition to the active compounds, may contain additionalagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Compositions of the Compound I or derivative thereof as described hereinfor rectal administrations are optionally suppositories, which can beprepared by mixing the compounds with suitable non-irritating excipientsor carriers such as cocoa butter, polyethyleneglycol or a suppositorywax, which are solid at ordinary temperatures but liquid at bodytemperature and therefore, melt in the rectum or vaginal cavity andrelease the active component.

Dosage forms for topical administration of the compounds describedherein or derivatives thereof include ointments, powders, sprays, andinhalants. The compounds described herein or derivatives thereof areadmixed under sterile conditions with a physiologically acceptablecarrier and any preservatives, buffers, or propellants as may berequired. Ophthalmic formulations, ointments, powders, and solutions arealso contemplated as being within the scope of the compositions.

The term pharmaceutically acceptable salt as used herein refers to thosesalts of Compound I or derivative thereof as described herein that are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of subjects without undue toxicity, irritation,allergic response, and the like, commensurate with a reasonablebenefit/risk ratio, and effective for their intended use, as well as thezwitterionic forms, where possible, of the compounds described herein.The term salts refers to the relatively non-toxic, inorganic and organicacid addition salts of Compound I or derivative thereof as describedherein. These salts can be prepared in situ during the isolation andpurification of the compounds or by separately reacting the purifiedcompound in its free base form with a suitable organic or inorganic acidand isolating the salt thus formed. Representative salts include thehydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate,oxalate, valerate, oleate, palmitate, stearate, laurate, borate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate,methanesulfonate, and laurylsulfonate salts, and the like. These mayinclude cations based on the alkali and alkaline earth metals, such assodium, lithium, potassium, calcium, magnesium, and the like, as well asnon-toxic ammonium, quaternary ammonium, and amine cations including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. (See Stahl and Wermuth, Pharmaceutical Salts: Properties,Selection, and Use, Wiley VCH, 2008, which is incorporated herein byreference in its entirety, at least, for the compositions taughttherein.)

The compounds described above or derivatives thereof are useful intreating or reducing the risk of disorders associated with activation ofthe TrkB receptor including neurological disorders, neuropsychiatricdisorders, and metabolic disorders (e.g., obesity). As used herein, theterms treating (including treat and treatment) or reducing the risk ofinclude prevention; delay in onset; diminution, eradication, or delay inexacerbation of one or more signs or symptoms after onset; andprevention of relapse.

Further, the compounds described above or derivatives thereof are usefulfor promoting neuroprotection in humans, e.g., including pediatric andgeriatric populations, and in animals, e.g., veterinary applications. Asubject in need of neuroprotection is a subject at risk for or having aneurological or neuropsychiatric disorder. Neurological orneuropsychiatric disorders include, for example, depression, anxiety,amyotrophic later sclerosis, Alzheimer's disease, Huntington's disease,Rett syndrome, epilepsy, Parkinson's disease, dementia, diabeticneuropathy, peripheral neuropathy, and central nervous system injuries.Central nervous system injuries include, for example, spinal cordinjury, stroke, hypoxia, ischemia, and brain injury.

The methods and compounds as described herein are useful for bothprophylactic and therapeutic treatment. For prophylactic use, atherapeutically effective amount of Compound I or derivative thereof asdescribed herein are administered to a subject prior to onset (e.g.,before obvious signs of neurological or neuropsychiatric disorder),during early onset (e.g., upon initial signs and symptoms ofneurological disorder), or after an established neurological disorder.Prophylactic administration can occur for several days to years prior tothe manifestation of symptoms of a disorder, e.g., a neurological or aneuropsychiatric disorder. Prophylactic administration can be used, forexample, in the preventative treatment of subjects diagnosed withgenetic predisposition or after onset of genetic neurological disorderssuch as Huntington's disease or prior to surgery in which stroke,hypoxia, or central nervous system injury is a risk. Therapeutictreatment involves administering to a subject a therapeuticallyeffective amount of Compound I or derivative thereof as described hereinafter a disorder, e.g., a neurological disorder, neuropsychiatricdisorder, or metabolic disorder (e.g., obesity), is diagnosed.

Administration of Compound I or derivative thereof as described hereincan be carried out using therapeutically effective amounts of Compound Ior derivative thereof as described herein for periods of time effectiveto treat a disorder. The effective amount of Compound I or derivativethereof as described herein may be determined by one of ordinary skillin the art and includes exemplary dosage amounts for a mammal of fromabout 0.5 to about 200 mg/kg of body weight of active compound per day,which may be administered in a single dose or in the form of individualdivided doses, such as from 1 to 4 times per day. Alternatively, thedosage amount can be from about 0.5 to about 150 mg/kg of body weight ofactive compound per day, about 0.5 to 100 mg/kg of body weight of activecompound per day, about 0.5 to about 75 mg/kg of body weight of activecompound per day, about 1 to about 70 mg/kg of body weight of activecompound per day, about 5 to about 60 mg/kg of body weight of activecompound per day, about 10 to about 60 mg/kg of body weight of activecompound per day, about 20 to about 60 mg/kg of body weight of activecompound per day, about 20 to about 50 mg/kg of body weight of activecompound per day, about 50 mg/kg of body weight of active compound perday, about 40 mg/kg of body weight of active compound per day, about 30mg/kg of body weight of active compound per day, about 20 mg/kg of bodyweight of active compound per day, about 10 mg/kg of body weight ofactive compound per day, or about 5 mg/kg of body weight of activecompound per day. Those of skill in the art will understand that thespecific dose level and frequency of dosage for any particular subjectmay be varied and will depend upon a variety of factors, including theactivity of the specific compound employed, the metabolic stability andlength of action of that compound, the species, age, body weight,general health, sex and diet of the subject, the mode and time ofadministration, rate of excretion, drug combination, and severity of theparticular condition.

In these methods, the disorder being treated, e.g., depression, anxiety,central nervous system injury, metabolic disorder (e.g., obesity), orother disorder, can be further treated with one or more additionalagents (e.g., an antidepressant, an anti-anxiolytic, an antiviral, or anantibiotic). The one or more additional agents and Compound I orderivative thereof as described herein can be administered in any order,including simultaneous administration, as well as temporally spacedorder of up to several days apart. The methods may also include morethan a single administration of the one or more additional agents and/orCompound I or derivative thereof as described herein. The administrationof the one or more additional agents and Compound I or derivativethereof as described herein may be by the same or different routes andconcurrently or sequentially. When treating with one or more additionalagents, the Compound I or derivative thereof as described herein can becombined into a pharmaceutical composition with the one or moreadditional agents. For example, Compound I or derivative thereof asdescribed herein can be combined into a pharmaceutical composition withan anti-depressant, such as, for example imipramine, fluoxetine,paroxetine, and/or sertraline. As a further example, Compound I orderivative thereof as described herein can be combined into apharmaceutical composition with an anti-anxiolytic, such as, for examplediazepam, alprazolam, clonazepam, and/or hydroxyzine.

The examples below are intended to further illustrate certain aspects ofthe methods and compounds described herein, and are not intended tolimit the scope of the claims.

EXAMPLES General Methods Cells, Reagents, and Mice

Human embryonic kidney 293 (HEK 293) cells were maintained in medium A(DMEM with 10% fetal bovine serum (FBS) and 100 units ofpenicillin-streptomycin), and DAT (dopamine transporter) stabletransfected HEK293 cells were cultured in DMEM with high glucose andL-glutamine (Lonza BioProducts; Basel, Switzerland) containing 10% FBSand 10 U/mL Pen/Strep with 0.4 mg/ml of G418 at 37° C. with 5% CO₂atmosphere in a humidified incubator. Brain-derived neurotrophic factor(BDNF) was obtained from Peptron (Santa Clara, Calif.). Phospho-Akt-473or 308 and Akt antibodies were from Cell Signaling (Danvers, Mass.).Anti-phospho-Erk1/2, anti-phospho-TrkA Y490, and TrkA and TrkBantibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz,Calif.). Anti-TrkB antibody was from Biovision (Mountain View, Calif.).Anti-p-TrkA 794 and anti-p-TrkB 816 has been previously described(Rajagopal et al., J. Neurosci., 24:6650-8, 2004; Arevalo et al., Mol.Cell. Biol. 20:5908-16, 2000, which are incorporated herein and in theirentirety at least with respect to these antibodies).

[³H]-dopamine, -norepinephrine, and -epinephrine were purchased from NewEngland Nuclear (Boston, Mass.). TrkA^(F592A) and TrkB^(F616A) mice havebeen described previously (Chen et al., Neuron, 46:13-21, 2005).TrkA^(F592A) and TrkB^(F616A) mice, TrkA +/−, TrkB +/−, TrkC +/−, NET−/− and BDNF +/− C57BL/6 mice were bred in a pathogen-free environmentin accordance with Emory Medical School guidelines. Unless otherwisenoted, chemicals were purchased from Sigma Aldrich (St. Louis, Mo.).

Cortex-Specific BDNF Deletion

The Cortex-Specific Cre mouse line was previously described as“transgenic line C” (Chhatwal et al., Gene Ther., 14:575-583, 2007,which is incorporated herein and in its entirety at least with respectto this mouse line). Briefly, coding sequence for Cre-recombinase(Cre-IRES-DsRed2) was placed downstream of a 3 kb cholecystokinin (CCK)promoter, linearized, purified, and microinjected into the pronuclei ofone-cell C57/BL6 embryos, which were then implanted into pseudo-pregnantC57/BL6 females. Following verification of gene expression in thedifferent transgenic lines, the cortex-specific “line C” was crossed toa floxed-stop lacZ reporter mouse line as well as a floxed BDNF mouseline (Chhatwal et al., Nat. Neurosci., 9:870-872, 2006; Soriano, Nat.Genet., 21:70-71, 1999; Rios et al., Mol. Endocrinol., 15:1748-1757,2001). Region specific Cre gene expression and BDNF deletion wereconfirmed with in situ hybridization, x-gal staining forbeta-galactosidase expression, and Western blot for BDNF protein levels.

Primary Rat Cortical and Hippocampal Neuron Cultures

Primary cultured rat cortical neurons were prepared as follows. E17 ratpups were decapitated and cortex was extirpated, cross chopped andsuspended by pipetting for separation in 5% fetal calf serum (FCS), 5%horse serum (HS) DMEM. The cell suspension was then centrifuged at 250×gfor 5 min. This operation was repeated. Cells were seeded intopolyethyleneimine-coated 10 cm² dishes and 12-well plates includingcoated-coverslips and incubated at 37° C. in 5% CO₂/95% air. After 3hours, the culture medium was changed to Neurobasal containing B-27supplement (Invitrogen; Carlsbad, Calif.) and incubated for 4 days. Formaintenance, half of the culture medium was changed to freshNeurobasal/B27 in every 4 days. After 1 week, the dished culturedneurons were ready for use.

Binding Constant Determination

Purified TrkA and TrkB ECD or ICD proteins were incubated with different³H-catecholamines at 25° C. for 30 minutes in 1 ml of binding buffer (50mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 nM ³H-catecholamines (32000 cpm)).After the incubation, the reaction mixture was loaded on a Whatmanglass-fiber filter (GF-B) (Whatman Inc.; Piscataway, N.J.). The mixturewas washed with 3×5 ml washing buffer (50 mM Tris-HCl, pH 7.4). Thedried filter paper was put into a small vial and subjected to liquidscintillation counter analysis. The value of the dissociate constant andthe number of sites were obtained from Scatchard plots by using theequation r/[L]free=n/Kd−r/Kd, where r is the ratio of the concentrationof bound ligand to the total protein concentration and n is the numberof binding sites.

Catecholamine Uptake in Cultured HEK-293 Cells and Rat Cortical Neurons

Uptake was performed on cultured cells in buffer containing 4 mM TrisBase, 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgSO₄, 5.6 mM glucose,6.25 mM HEPES, pH to 7.4 with KOH, 1 mM Ascorbate, and 100 nM pargyline(Sigma Aldrich, St. Louis, Mo.). For dopamine (DA) uptake, the cellswere incubated for 2 minutes in 1 μM DA (Sigma Aldrich; St. Louis, Mo.)with a 2% tracer of ³H-DA (Perkin Elmer; Boston, Mass.). For nonspecificuptake measures, the cells were preincubated with the respectiveinhibitor for 15 minutes. After uptake, the cells were washed twice withice-cold buffer to stop the uptake. Cells were then lysed and harvestedin 0.1N NaOH. Lysates were added to ScintSafe Econo 1 scintillationfluid (Fisher Scientific; Pittsburgh, Pa.) and allowed to standovernight in the dark. The subsequent day, samples were read in a LS6500Beckman Scintillation Counter (Beckman Coulter, Inc.; Fullerton,Calif.). Specific uptake measurements were calculated by subtractingnon-specific uptake from total uptake.

Immunofluorescent Staining

Primary hippocampal neurons were seeded on poly-L-lysine coatedcoverslips in a 12-well plate. After 7 days in vitro, the neurons weretreated with 100 ng/ml BDNF or variety of neurotransmitters and7,8-dihydroxyflavone compound (500 nM) for 30 min, and then washed withPBS. Cells were fixed with 3% formaldehyde in PBS at room temperaturefor 15 min. The cells were then permeabilized and blocked by 0.4% TritonX-100 and 2% FBS in PBS at room temperature for 15 minutes, washed withPBS three times and treated with anti-MAP2 (1:200) and anti-phospho-TrkBantibodies (1:100). After staining with FITC- or Rhodamine-conjugatedsecondary antibody, the coverslips were mounted on slides. Fluorescentimages were taken by OLYMPUS IX71 fluorescence microscope (Olympus;Center Valley, Pa.).

TrkB F616A Mice Treatment with L-DOPA and DOPS

The DOPS concentration was 20 mg/ml, the vitamin C concentration was 2mg/ml, and the benserazide concentration was 5 mg/ml. The vitamin Cprevents oxidation of the DOPS, and the benserazide is a peripheralinhibitor of AADC and restricts the production of NE from DOPS to thebrain. The compounds were added to a tube, and 20 μl of 10 M HCl wasadded to each ml of DOPS solution. The appropriate amount of water wasadded to the tube, and then HCl was added to achieve the desired volume.For example, to make 1 mL of a DOPS solution, 20 mg DOPS, 2 mg vitaminC, and 5 mg benserazide were added to a tube, followed by 980 μL ofwater and 20 μL of 10 M HCl. The solution was vortexed until all DOPSwas dissolved. The DOPS solution was injected subcutaneously in a volumeof 50 μl/g of mouse. The appropriate amount of DOPS solution waspipetted into a clean tube, followed by the amount of 10 M NaOH requiredto exactly neutralize the HCl. The solution was then shaken. The amountof 10 M NaOH is the weight of the mouse in microliters. For example, fora 20 g mouse, pipet 1 ml of the DOPS solution is pipetted into a tubeand 20 μL of 10 M NaOH is added. For a 30 g mouse, 1.5 mL of the DOPSsolution is piepetted into a tube and 30 μl of 10 M NaOH is added. Twoto four month-old TrkBF616A mice were pretreated with 1NMPP1 in drinkingwater (50 μM) 1 day before L-DOPA (50 mg/kg) or DOPS (100 mg/kg)intraperitoneal injection. At times of 2 hours and 5 hours after drugadministration, the L-DOPA or DOPS-treated mice were sacrificed,respectively. The brain lysates were prepared and analyzed byimmunoblotting against anti-phospho-TrkB Y816 and TrkA Y794 antibodies.

Example 1 Catecholamines Induce TrkB Activation in Primary Neurons in aDose-Dependent Manner

To explore whether monoamine neurotransmitters, including catecholamines(see, e.g., FIG. 1), could stimulate TrkB activation, hippocampalneurons were treated with 300 nM of catecholamines, serotonin, andmelatonin for 30 min. Immunofluorescent staining with anti-phospho-TrkBY816 showed that catecholamines, like BDNF, strongly triggered TrkBtyrosine phosphorylation, whereas serotonin and melatonin did not (FIG.2). Other serotonin metabolites, including 5-HIAA (5-hydroxyindoleaceticacid) and 5-HT-sulfate, had no effect either. Immunoblotting analysiswith the neuronal lysates revealed that TrkB, but not TrkA, wasselectively activated by catecholamines. The analysis also showed thatTrkB was not activated by serotonin or melatonin. The downstreamsignaling activation, including Akt and MAPK, correlates with TrkBphosphorylation (FIG. 3). K252a is a selective inhibitor of the tyrosinekinase activity of the Trk family of neurotrophin receptors. K252apretreatment robustly blocked catecholamine-triggered TrkB tyrosinephosphorylation (FIG. 4), indicating that the stimulatory effect bycatecholamines represented Trk receptor-dependent autophosphorylation.To gain the full spectrum of catecholamines' dosage in activating TrkB,cortical neurons were treated with different doses of dopamine,norepinephrine, and epinephrine for 30 min. Catecholamines induced TrkBactivation in a dose dependent manner, and demonstrable TrkBphosphorylation was detected when the concentration of catecholamine wasas low as 10 nM (FIG. 5). Time course experiments showed thatcatecholamine-induced TrkB activation started at 10 minutes, peaked at15 minutes, and sustained at 30-60 minutes, in a kinetic pattern similarto BDNF. Therefore, in the following experiments, catecholaminestimulation time point was set at 15 minutes (FIG. 6). Thus, these datademonstrated that catecholamines, but not serotonin or melatonin,rapidly caused TrkB activation in primary neurons.

Example 2 Catecholamines Directly Bind the Intracellular Domain of TrkBand Trigger its Dimerization

To determine whether catecholamines bind TrkB directly, an in vitrobinding assay was conducted with purified ECD (extracellular domain) andICD (intracellular domain) proteins from Trk receptors and³H-catecholamines. The ICD, but not ECD, from the TrkB receptor bound tocatecholamines. Epinephrine exhibited the strongest binding activity. Bycontrast, the counterparts of TrkA did not bind to ³H-catecholamines. Asa positive control, 7,8-dihydroxyflavone selectively bound to the ECD ofTrkB. Quantitative analysis by Scatchard plot revealed that the TrkBbinding constants by dopamine, norepinephrine, and epinephrine were 76μM, 98 μM, and 1.1 μM, respectively (FIGS. 7A & B). To determine whetherthe binding by catecholamines to TrkB receptor caused dimerization,GST-TrkB was co-transfected into HEK293 cells with HA-TrkB, followed byBDNF or monoamines treatment for 15 minutes. Co-immunoprecipitationdemonstrated that catecholamines, but not serotonin or melatonin,elicited TrkB homodimerization as BDNF (FIG. 8, top panel). Epinephrineelicited tyrosine phosphorylation in TrkB, but not in TrkA or TrkCreceptors in transfected HEK293 cells (FIG. 9). In contrast, TrkB-KD(kinase-dead) receptors were not tyrosine phosphorylated. A similarobservation was made with dopamine and norepinephrine. These dataindicated that tyrosine phosphorylation of TrkB receptor provoked bycatecholamines is exerted by the receptor autophosphorylation but not byany other cytoplasmic tyrosine kinases. Hence, catecholaminesselectively induced TrkB dimerization and tyrosine autophosphorylation.

To test whether expression of catecholamine transporters enhanced TrkBactiviation by extracellular catecholamines, HEK293 cells weretransfected with DAT (dopamine transporter) or NET (norepinephrinetransporter) and treated with various amounts of catecholamines for 5minutes. In control cells, the minimal required catecholamine toactivate transfected TrkB was 100 nM, where 10 nM dopamine wassufficient to activate TrkB in DAT transfected cells. Inclusion of NETin HEK293 cells also substantially enhanced TrkB activation bynorepinephrine or epinephrine. Overexpression of NET increased thestimulatory activity approximately 100 fold as compared to controlcells, supporting that catecholamine transporters facilitatecatecholamines-mediated TrkB activation.

Example 3 Catecholamines Specifically Activate TrkB in NeurotrophinsIndependent Manner

Both antidepressants and exercise increase hippocampal BDNF mRNAexpression through enhanced 5-HT and/or NE neurotransmission (Siuciak etal., Pharmacol. Biochem. Behav., 56:131-137, 1997; Dias et al.,Neuropharmacology, 45:553-563, 2003; Ivy et al., Pharmacol. Biochem.Behav., 75:81-88, 2003; Garza et al., Pharmacol. Biochem. Behav.,77:209-220, 2004). If catecholamines activated TrkB signaling indirectlyby promoting BDNF generation, then scavenging BDNF with its specificantibody would be expected to diminish catecholamines-mediatedactivation of TrkB. Cultured cortical neurons were pretreated withBDNF-IgG for 30 minutes followed by exposure to BDNF (10 ng/ml) orcatecholamines (100 nM) for 15 minutes. Pretreatment with BDNF-IgGabolished BDNF-induced phosphorylation of TrkB; by contrast, BDNF-IgGhad no effect on 7,8-dihydroxyflavone or catecholamine-inducedphosphorylation of TrkB (FIG. 10), suggesting that the action ofcatecholamines is independent of BDNF. Likewise, addition of either NT-3or NT-4 antibody or their combination to primary neuronal culturesfailed to prevent or reduce catecholamines-mediated activation of TrkB(FIG. 11). It was then determined whether catecholamines could activateTrkB in neurons cultured from mice carrying a null mutation of BDNF.Addition of catecholamines for 15 min to cortical neurons cultured fromBDNF-null mice resulted in evident activation of TrkB as BDNF and7,8-dihydroxyflavone, whereas serotonin had no effect (FIG. 12).

To further investigate the specificity of catecholamines in inducingTrkB activation, cortical neurons were pretreated with control IgG ornorepinephrine IgG, a rabbit polyclonal antibody for norepinephrine,followed by catecholamines. Compared to control IgG, norepinephrine IgGselectively neutralized the agonistic effect of norepinephrine, but notdopamine or epinephrine (FIG. 13). To examine whether the TrkBstimulatory effect by catecholamines is exerted through indirectactivation of TrkB by traditional catecholamine receptors, corticalneurons were pretreated with various pharmacological antagonists todopamine and norepinephrine receptors. Then 100 nM dopamine ornorepinephrine was added, respectively. Blockade of dopamine ornorepinephrine receptors failed to abrogate TrkB activation by thecatecholamines (FIG. 14), indicating that catecholamines do not causeTrkB activation via their G-protein coupled receptors. Therefore, thesedata support that catecholamines trigger TrkB activation in acatecholamine G-protein receptor independent manner.

Example 4 Catecholamines Selectively Activate TrkB Receptor in Mice

To determine if catecholamines can selectively activate TrkB receptor,cortical neurons were prepared from pups of TrkB +/− mice, which weremated to mice of the same genotype. Catecholamines, but not serotonin,specifically activated TrkB in wild-type but not TrkB—null neurons,whereas TrkA was not activated in either neuron (FIG. 15A, top and3^(rd) panels). Moreover, catecholamines strongly provoked TrkB but notTrkA activation in both wild-type and TrkC knockout neurons (FIG. 15B,top and 3^(rd) panel). To further explore whether catecholamines canprovoke TrkB activation in vivo, TrkB F616A knock-in mice were employed,where TrkB F616A can be selectively blocked by 1NMPP1 inhibitor and leadto TrkB-null phenotypes.

To assess whether catecholamines can mimic BDNF, cortical neurons wereprepared from TrkB F616A knock-in mice and were pretreated withinhibitors K252a or 1NMPP1. As described in Example 1, K252a is a waterinsoluble, selective inhibitor of the tyrosine kinase activity of theTrk family of neurotrophin receptors, including wild-type TrkB. 1NMPP1is a water-soluble derivative of K252a and functions as selectiveinhibitor of mutated TrkB receptors, such as TrkB F616A, but notwild-type TrkB. Catecholamine-provoked TrkB phosphorylation wasselectively reduced by 1NMPP1 but not K252a, whereas serotonin had noeffect. As a control, TrkA was not activated (FIG. 16, top and 3^(rd)panels). Catecholamine-induced TrkB activation in TrkA F592A neurons wasrobustly inhibited by K252a but not by 1NMPP1. These findings suggestthat catecholamines strongly provoked both wild-type TrkB and TrkB F616Atyrosine phosphorylation and activation. Because 1NMPP1 selectivelyinhibits TrkB F616A activation by catecholamines in vitro, 1NMPP1 mayalso block TrkBF616A activation by catecholamines in mice. L-DOPA(L-dihydroxyphenylalanine, 50 mg/kg) and DOPS (dihydroxyphenylserine,100 mg/kg), precursors of dopamine and norepinephrine, respectively,were employed, because dopamine or norepinephrine can not pass throughthe brain blood barrier. TrkB F616A mice were pretreated with 1NMPP1 (50μM) before L-DOPA or DOPS administration. As a control, saline and1NMPP1 had no effect on TrkB tyrosine phosphorylation in mice. As apositive control, 7,8-dihydroxyflavone, L-DOPA and DOPS provoked evidentTrkB activation, and pretreatment of 1NMPP1 markedly diminished TrkBactivation in F616A mice. Compared to vehicle control, administration ofL-DOPA or DOPS into BDNF conditional knockout mice strongly triggeredTrkB activation in BDNF −/− cortex. Thus, catecholamines and theirprecursors strongly and selectively activated TrkB in mice.

Mice lacking dopamine β-hydroxylase (DBH) are unable to synthesizenoradrenaline (essential for mouse fetal development) or adrenaline anddie in utero. Administration of DOPS, which can be converted tonoradrenaline in the absence of DBH, rescued DBH −/− mice from dying.Mature DBH −/− mice can live without supplemental of DOPS. To explorewhether exogenously administrated DOPS, which can be changed intonorepinephrine and epinephrine, induced TrkB activation in DBH −/− mice,mice were intraperitoneally injected with DOPS and TrkB activation wasmonitored at different time courses. In wild-type control mice, DOPSinduced TrkB activation in a time dependent manner with the maximaleffect at 12 hours. In contrast, DOPS-triggered TrkB activation occurredat 1 hour, peaked at 5 hours, and decreased at 12 hours in DBH −/− mice.As a control, TrkA was not activated. Quantitative analysis revealedthat BDNF in mouse brain was not significantly altered during the drugtreatment. Moreover, L-DOPA and DOPS rapidly induced TrkB activation inprimary cultured neurons, indicating that L-DOPA and DOPS directlyactivate TrkB like catecholamines. Hence, these data demonstrated thatcatecholamines selectively activate TrkB receptor in mice.

Example 5 Neurotransmitter Transporters Regulate TrkB Activation byCatecholamines

To test whether catecholamine transporters are involved in TrkBactivation by extracellular catecholamines, cortical neurons werepretreated with various DAT, NET or OCT3 (organic cation transporter 3)inhibitors, followed by dopamine, norepinephrine, and epinephrinetreatment. DAT inhibitors GBR 12909 and GBR12935, but not NET inhibitorsdesipramine or nisoxetine, significantly blocked dopamine-mediated TrkBactivation. On the other hand, norepinephrine and epinephrine-provokedTrkB activation was selectively decreased by NET inhibitors but not OCT3or DAT inhibitors (FIG. 17). Further, the combination of DAT and NETinhibitors robustly repressed TrkB activation in cortical neurons bydopamine, norepinephrine, and epinephrine (FIG. 18), underscoring thatcatecholamines influx is involved in their agonistic effect on TrkB. Itwas then determined whether catecholamines could activate TrkB inneurons cultured from mice carrying a null mutation of NET. Addition ofdopamine into NET −/− neurons resulted in notable TrkB activationsimilar to that in wild-type control neurons, whereas norepinephrine orepinephrine-mediated TrkB activation was completely blocked in NET −/−neurons compared to control neurons (FIG. 19). Taken together, thesefindings demonstrate that catecholamines activating TrkB requiresintracellular locale mediated by transporters.

To determine whether the norepinephrine antibody selectively blocks theagonistic effect of catecholamines, rat cortical neurons were pretreatedfor 15 min with either the control IgG or norepinephrine IgG, a rabbitpolyclonal antibody for norepinephrine, followed by 300 nMcatecholamines for another 15 min. The cell lysates were analyzed byimmunoblotting with anti-p-TrkB-Y-816. Compared with control IgG,norepinephrine IgG selectively inhibited the agonistic effect ofnorepinephrine but not dopamine or epinephrine on TrkB activation (FIG.20).

To determine whether dopamine and adrenergic receptor antagonists blockthe agonistic effect of catecholamines on TrkB, rat cortical neuronswere pretreated with various pharmacological antagonists to dopamine andepinephrine receptors for 30 min, followed by 100 nM of dopamine orepinephrine treatment for 15 min, respectively. The pharmacologicalantagonists to dopamine receptors included SCH23390 (10 μM) andhaloperidol (300 nM). The pharmacological antagonists to epinephrinereceptors included prazosin (50 nM), yohimbine (10 μM), and propranolol(10 μM). The cell lysates were analyzed by immunoblotting withanti-p-TrkB-Y-816. Both the dopamine and norepinephrine receptorantagonists failed to affect the activation of TrkB by thecatecholamines (FIG. 21).

Example 6 OCT3 Inhibitor has No Effect on TrkB Activation byCatecholamines

HEK293 cells were transfected with TrkB and pretreated with the OCT3inhibitor decynium (2 μM) for 30 minutes, followed by stimulation withcatecholamines for 15 minutes. The cell lysates were monitored byimmunoblotting with anti-p-TrkB-Y-816. The OCT3 inhibitor failed toinhibit the TrkB activation by catecholamines (FIG. 22A, upper panel).An equal amount of TrkB was expressed in each sample (FIG. 22A, lowerpanel).

Example 7 EDTA Blocks the Stimulatory Effect on TrkB Activation by Znbut not Catecholamine

A group of primary cortical neurons were pretreated with 1 mM EDTA, andanother group of primary cortical neurons were not pretreated. Bothgroups of primary cortical neurons were then treated with either Zn (100μM) or a catecholamine (100 nM) for 15 minutes. The neuronal celllysates were analyzed by immunoblotting with anti-p-TrkB-Y-816 antibody.The stimulatory activity of TrkB activation by Zn, but notcatecholamines, was blocked by EDTA (FIG. 22B).

Example 8 Catecholamine Derivatives Orally Activate TrkB Receptor

Catecholamines can directly bind to the kinase domain of the TrkBreceptor and activate it. However, the catecholamines are not able tocross through the blood brain barrier. To search for catecholaminederivatives that can pass through the blood brain barrier via oraladministration, several compounds containing the catecholamine backbonedescribed herein (Compounds I-2, I-5, I-8, I-9, I-10, I-11, I-17, andI-18) were tested on primary cortical neurons. Cortical neurons (7 DIVcortical neurons from 18 day old Sprague Dawley rat embryos) weretreated with 500 nM of the selected compound for 30 min and cell lysateswere prepared. The cell lysates (20 mg) were analyzed for TrkBactivation by immunoblotting with anti-p-TrkB-Y-816 antibody. Among thetested compounds, Compound I-11 (2-amino-3′,4′-dihydroxypropiophenone)and Compound I-8 (α-dimethylamino-3′,4′-dihydroxyacetophenone)prominently activated TrkB (FIG. 23).

To explore whether these compounds could stimulate TrkB in vivo, 50mg/kg of Compound I-11, Compound I-8, and Compound I-2(2-(methylamino)-3′,4′-dihydroxy-acetophenone) were each orallyadministered (in saline) to 2-month-old C57BL/6J mice. The mice weresacrificed at different time points and TrkB activation in the brains ofthe mice was monitored by immunoblotting with anti-p-TrkB-Y-816antibody. Compound I-11 and Compound I-8 potently activated TrkB 1 to 2hours after oral administration. The activity escalated with the timecourse and peaked at 8 hours, suggesting that both compounds are orallybioactive. In contrast, Compound I-2 failed to activate TrkB (FIGS. 24A,24B, & 24C).

To determine the minimal required dosages for Compound I-11 and CompoundI-8, different doses of the compounds were each orally administered tomice. The mice were sacrificed after 4 hours, and the brain lysates wereanalyzed by immunoblotting with anti-p-TrkB-Y-816 antibody. A titrationassay demonstrated that the minimal dosages required for Compound I-11and Compound I-8 to activate TrkB were 10 mg/kg and 20 mg/kg (FIGS. 25A& 25B). These results indicate that orally administered Compound I-11and Compound I-8 can pass through the blood brain barrier and activateTrkB receptor in mouse brain.

Example 9 Screening for Catecholamine Derivatives that can Activate TrkBReceptor in Primary Neurons

Catecholamine derivatives as described herein, including Compound I-2,Compound I-5, Compound I-7, Compound I-8, Compound I-9, Compound I-10,Compound I-11, Compound I-14, Compound I-15, Compound I-17, CompoundI-18, and isoproterenol, were screened for TrkB activation in primarycortical neurons. Cortical neurons (7 DIV cortical neurons from 18 dayold Sprague Dawley rat embryos) were treated with 500 nM of the selectedcompound for 30 min and cell lysates were prepared. The cell lysates (20mg) were analyzed for TrkB activation by immunoblotting withanti-p-TrkB-Y-816 antibody. The results showed that Compound I-11(2-amino-3′,4′-dihydroxypropiophenone, ADPP) and Compound I-8(dimethylamino-3′,4′-dihydroxyacetophenone, DDAP) displayed prominentstimulatory effect on the TrkB receptor (FIGS. 26A and 26B). As shown inFIG. 26B, Compound I-2, Compound I-7, and Compound I-15 displayactivity. Taken together, these data support that Compound I-8 andCompound I-11 possess robust TrkB stimulatory effect.

Example 10 Development of Orally Bioactive Catecholamine Derivatives forActivation of TrkB

To search for orally bioactive catecholamines, catecholamine derivativesdescribed herein were screened using primary neuronal cultures. The invitro active compounds, Compounds I-8 and I-11, were adminstered intotwo to three month old C57BL/6J mice orally, intraperitoneally, and indrinking water. For oral administration, the mice were administered 0 mg(control), 5 mg, 10 mg, 20 mg, or 50 mg of Compound I-8 or CompoundI-11. After 4 hours, TrkB phosphorylation in mouse brain was monitoredby analyzing the brain lysates using immunoblotting withanti-p-TrkB-Y-816 antibody. The ratios of p-TrK/total TrkB werequantified (FIG. 27A). For intraperitoneal administration, 50 mg/kg ofCompound I-8 or Compound I-11 were intraperitoneally injected into themice. The brain lysates were prepared at different times (0 hr, 1 hr, 2hr, 4 hr, and 8 hr) and were analyzed by immunoblotting withanti-p-TrkB-Y-816 antibody (FIG. 27B). Compound I-8 and Compound I-11were dissolved in drinking water and given to mice. After 24 h, thebrain lysates were prepared and analyzed by immunoblotting withanti-p-TrkB-Y-816 antibody (FIG. 27C). Compounds I-8 and I-11 were bothshown to be orally bio-available catecholamine derivatives that potentlysuppress neuroexcitotoxin-provoked neuronal cell death in aTrkB-dependent manner. Moreover, these compounds potently activate TrkBin mouse brains when administrated via drinking water or intraperitonealinjection (FIGS. 27A-C). Therefore, these compounds are useful intreating various neurological or neuropsychiatric diseases as describedherein.

Example 11 Catecholamine Derivatives Suppress Neuronal Apoptosis

Norepinephrine has neuroprotective activity against oxidativestress-induced neuronal apoptosis. To investigate whether thisprotective action by norepinephrine is mediated through TrkB, apoptosisof TrkB F616A cortical neurons was monitored. Primary cortical neuronswere pretreated with inhibitors K252a or 1NMPP1 followed by overnighttreatment with 50 mM glutamate. As shown in FIG. 28A, norepinephrine(NE) robustly suppressed glutamate-provoked neuronal cell death and thisprotective action was abolished by 1NMPP1 (1NMPP1+NE) but not by K252a(K252a+NE)(apoptosis was quantified using the TUNEL assay). Hence, theneurotrophic activity of NE is TrkB receptor dependent.

To determine whether Compound I-11 (ADPP) and Compound I-8 (DDAP)protect neurons from KA-induced apoptosis, TrkB F616A knockin mice werepretreated with inhibitor 1NMPP1 (16.6 ng/kg) 2 days before theexperiment. Compound I-8 (DDAP) or Compound I-11 (ADPP) (50 mg/kg) wasorally injected into the mice 2 hours before kainic acid (KA)administration (20 mg/kg). Brain lysates were analyzed by immunoblottingand Caspase-3 ELISA. It was found that oral administration of CompoundsI-8 and I-11 robustly repressed kainic acid (KA)-provoked neuronal celldeath in mouse brain and 1NMPP1 pretreatment blocked the protectiveeffect (FIG. 28B, left panel). The extent of the protective effectcorrelates with TrkB activation status by these compounds. ActiveCaspase-3 ELISA correlated with the immunoblotting results (FIG. 28B,right panel).

To determine whether Compounds I-8 and I-11 suppress neuronal apoptosisin a TrkB dependent manner, primary cortical neurons were prepared fromTrkB F616A knockin mice. On day 7, the primary cortical neurons werepretreated with 100 nM K252a or 1NMPP1 for 30 min, followed by 500 nMADPP and DDAP. Cell lysates were analyzed by Western blotting. TrkBactivation by Compounds I-8 and I-11 in primary TrkB F616A corticalneurons was selectively blocked by 1NMPP1 but not K252a (FIG. 28C).These data demonstrate that catecholamine and its derivatives suppressneuronal cell death through activating TrkB.

Example 12 Catecholamine Derivatives Reveal Potent Antidepressant Effect

Accumulating evidence supports that BDNF plays an essential role inmediating antidepressants' therapeutic effects. Infusion of exogenousBDNF into hippocampus or brain stem has antidepressant-like behavioraleffect. A forced swim test is broadly used for screening of potentialantidepressant drugs and is widely used to measure antidepressantactivity. To explore whether Compound I-11 (ADPP) and Compound I-8(DDAP) have antidepressant effects like BDNF, forced swim tests wereconducted. Adult male mice (2-3 months old, n=8) were randomlysubmitted, without a pre-swim, to a forced swim test of 6 minutes withimmobility recorded in the last 4 minutes. Mice were injectedintraperitoneally with saline, Compound I-11 (20 mg/kg), or Compound I-8(20 mg/kg). The mice were allowed to adapt to the test room for 2 days,and the mice were placed in a clear glass cylinder with a diameter of 16cm, half-filled with clear water at 24° C. The water depth of 14 cm didnot allow the mice to reach the bottom of the cylinder, and the waterwas changed after each mouse. When the mice were treated with CompoundI-8 or Compound I-11 (20 mg/kg), the swimming immobility wassignificantly decreased (see FIG. 29A), suggesting that both compoundsimitate BDNF and exert potent anti-depressant effect.

To assess whether the behavior responses caused by catecholaminederivatives is mediated by TrkB receptor, TrkB F616A knockin mice wereutilized. The mice were subjected to saline or 1NMPP1 inhibitorpretreatment. No significant difference was observed in the immobilitytimes between the saline and 1NMPP1 treated control groups. In thesaline group, both compounds substantially reduced the immobility time;in contrast, Compound I-8 had no significant effect on the immobilitytime after 1NMPP1 treatment (FIG. 29B), suggesting that inhibition ofTrkB signaling cascade blocks the antidepressant effect by Compound I-8.Compound I-11 was still active in decreasing immobility even in thepresence of 1NMPP1. These data demonstrate that DDAP mimics BDNF andacts as a potent antidepressant drug in mice through activating TrkBreceptor.

The present compounds, compositions, and methods are not limited inscope by the examples described herein, which are intended asillustrations of a few aspects of the compounds, compositions, andmethods and any examples that are functionally equivalent are within thescope of the disclosure. Various modifications of the compounds,compositions, and methods in addition to those shown and describedherein are intended to fall within the scope of the appended claims.Further, while only certain representative compounds, compositions,methods, and aspects of these compounds, compositions, and methods arespecifically described, other compounds, compositions, and methods andcombinations of various features of the compounds, compositions, andmethods are intended to fall within the scope of the appended claims,even if not specifically recited. Thus a combination of steps, elements,components, or constituents may be explicitly mentioned herein; however,all other combinations of steps, elements, components, and constituentsare included, even though not explicitly stated.

This listing of claims will replace all prior versions, and listings, ofclaims in the application:

1. A compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: R¹ ishydrogen, —OH, or ═O; R² is hydrogen, substituted or unsubstituted C₁₋₄alkyl, substituted or unsubstituted C₁₋₄ heteroalkyl, substituted orunsubstituted C₂₋₄ alkenyl, substituted or unsubstituted C₂₋₄heteroalkenyl, substituted or unsubstituted C₂₋₄ alkynyl, substituted orunsubstituted C₂₋₄ heteroalkynyl, or substituted or unsubstitutedcarbonyl; R³ and R⁴ are each independently selected from hydrogen,substituted or unsubstituted C₁₋₁₂ alkyl, substituted or unsubstitutedC₃₋₁₂ cycloalkyl, substituted or unsubstituted C₁₋₁₂ heteroalkyl,substituted or unsubstituted C₂₋₁₂ alkenyl, substituted or unsubstitutedC₃₋₁₂ cycloalkenyl, substituted or unsubstituted C₂₋₁₂ heteroalkenyl,substituted or unsubstituted C₂₋₁₂ alkynyl, substituted or unsubstitutedC₃₋₁₂ cycloalkynyl, substituted or unsubstituted C₂₋₁₂ heteroalkynyl,substituted or unsubstituted carbonyl; and R⁵, R⁶, and R⁷ are eachindependently selected from hydrogen, halogen, —OH, or alkoxy, whereinone of R¹, R², R³, R⁴, R⁵, R⁶, or R⁷ is not hydrogen; wherein if one ofR³ or R⁴ is a saccharide, R⁵ is not hydrogen; and wherein if R¹ is —OH,one of R², R³, R⁴, R⁵, R⁶, or R⁷ is not hydrogen; if R¹ is —OH and oneof R³ or R⁴ is —CH₃ or isopropyl, one of R², R³, R⁴, R⁵, R⁶, or R⁷ isnot hydrogen; if R² is —COOH, one of R¹, R³, R⁴, R⁵, R⁶, or R⁷ is nothydrogen; and if R² is —COOH and R¹ is —OH, then one of R³, R⁴, R⁵, R⁶,or R⁷ is not hydrogen.
 2. The compound of claim 1, wherein R² and NR⁴are combined to form a substituted or unsubstituted heterocycloalkyl orsubstituted or unsubstituted heteroaryl, wherein the heteroaryl is notsubstituted or unsubstituted pyrrole.
 3. The compound of claim 1,wherein R³ and NR⁴ are combined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl.
 4. Thecompound of claim 1, wherein the halogen is fluorine.
 5. The compound ofclaim 1, wherein R² is

wherein R⁸ and R⁹ are each independently selected from hydrogen,substituted or unsubstituted C₁₋₄ alkyl, substituted or unsubstitutedC₂₋₄ alkenyl, or substituted or unsubstituted C₂₋₄ alkynyl.
 6. Thecompound of claim 1, wherein R² is


7. The compound of claim 1, wherein the compound is


8. The compound of claim 1, wherein the compound is


9. A method of treating or reducing the risk of depression, anxiety, orobesity in a subject, comprising administering to the subject atherapeutically effective amount of a compound of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: R¹ ishydrogen, —OH, or ═O; R² is hydrogen, substituted or unsubstituted C₁₋₄alkyl, substituted or unsubstituted C₁₋₄ heteroalkyl, substituted orunsubstituted C₂₋₄ alkenyl, substituted or unsubstituted C₂₋₄heteroalkenyl, substituted or unsubstituted C₂₋₄ alkynyl, substituted orunsubstituted C₂₋₄ heteroalkynyl, or substituted or unsubstitutedcarbonyl; R³ and R⁴ are each independently selected from hydrogen,substituted or unsubstituted C₁₋₁₂ alkyl, substituted or unsubstitutedC₃₋₁₂ cycloalkyl, substituted or unsubstituted C₁₋₁₂ heteroalkyl,substituted or unsubstituted C₂₋₁₂ alkenyl, substituted or unsubstitutedC₃₋₁₂ cycloalkenyl, substituted or unsubstituted C₂₋₁₂ heteroalkenyl,substituted or unsubstituted C₂₋₁₂ alkynyl, substituted or unsubstitutedC₃₋₁₂ cycloalkynyl, substituted or unsubstituted C₂₋₁₂ heteroalkynyl,substituted or unsubstituted carbonyl; and R⁵, R⁶, and R⁷ are eachindependently selected from hydrogen, halogen, —OH, or alkoxy, whereinone of R¹, R², R³, R⁴, R⁵, R⁶, or R⁷ is not hydrogen; wherein if one ofR³ or R⁴ is a saccharide, R⁵ is not hydrogen; and wherein if R¹ is —OH,one of R², R³, R⁴, R⁵, R⁶, or R⁷ is not hydrogen; if R¹ is —OH and oneof R³ or R⁴ is —CH₃, one of R², R³, R⁴, R⁵, R⁶, or R⁷ is not hydrogen;if R² is —COOH, one of R¹, R³, R⁴, R⁵, R⁶, or R⁷ is not hydrogen; and ifR² is —COOH and R¹ is —OH, then one of R³, R⁴, R⁵, R⁶, or R⁷ is nothydrogen.
 10. The method of claim 9, further comprising selecting asubject with or at risk of developing depression, anxiety, or obesity.11. A method of promoting neuroprotection in a subject, comprisingadministering to the subject a therapeutically effective amount of thecompound of claim 1 or derivative thereof.
 12. (canceled)
 13. The methodof claim 11, wherein the subject has amyotrophic lateral sclerosis. 14.The method of claim 11, wherein the subject has a central nervous systeminjury.
 15. The method of claim 14, wherein the central nervous systeminjury is a brain injury.
 16. The method of claim 14, wherein thecentral nervous system injury is a spinal cord injury.
 17. The method ofclaim 14, wherein the central nervous system injury is a stroke. 18-30.(canceled)